The therascreen PITX2 RGQ PCR Kit is a CE-IVD kit for determination of the percent methylation ratio (PMR) in promoter 2 of the pituitary homeobox transcription factor 2 (PITX2) gene using methylation-specific real-time PCR on the Rotor-Gene Q 5-plex HRM instrument. The kit provides reagents optimized for reliable and sensitive determination of the PITX2 methylation status, and allows for a clinically validated determination of the PMR cut-off value of 12, which is predictive of patient response to anthracycline-based chemotherapy with or without endocrine therapy when used to test formalin-fixed, paraffin-embedded (FFPE) tissue samples from lymph node-positive, estrogen receptor-positive and HER2-negative high-risk breast cancer patients.

IMPORTANT: Does not include license for use. License key must be purchased separately.

Visit the Download Page for download, installation and licensing instructions.

The therascreen PITX2 RGQ PCR Kit is intended for in vitro diagnostic use in Europe.

Performance

To ensure reliable and sensitive determination of the PITX2 promoter 2 methylation status, the therascreen PITX2 RGQ PCR Kit has been optimized to detect and differentiate between a methylated and unmethylated state in the target region of the PITX2 promoter 2. For samples with a low PMR, the limit of detection (LOD) is 4 and for samples with a high PMR the upper detection limit is 92, facilitating a highly reliable determination of PMR values in the range in-between. Additionally, the assay has a wide range of linearity, from PMR 5 to 50. The clinical validity of the pre-defined PMR cut-off of 12 to predict patient response to anthracycline-based chemotherapy with or without endocrine therapy was shown by using 145 patient samples with 10-year follow-up data and disease-free survival as the primary end point.

Principle

The therascreen PITX2 RGQ PCR Kit exploits the real-time PCR oligonucleotide hydrolysis principle of TaqMan probes in combination with methylation-unspecific primers. Distinction between methylated and unmethylated status of the amplified bisulfite-converted target sequence in the PITX2 promoter 2 is achieved by using the combination of a FAM-labeled probe hybridizing specifically to the methylated bisulfite-converted DNA sequence and a HEX-labeled probe specific for the unmethylated bisulfite-converted DNA sequence. The resulting FAM- and HEX-specific Ct values are then used for the automated calculation of the PMR.

Procedure

The first step is manual DNA extraction using the QIAamp DSP DNA FFPE Tissue Kit, followed by quantification of genomic DNA and then bisulfite conversion with EpiTect Fast DNA Bisulfite Kit. Bisulfite-converted DNA is then amplified by real-time PCR, with all reagents provided in the therascreen PITX2 RGQ PCR Kit and the methylation status of the PITX2 promoter 2 is determined using the Rotor-Gene Q MDx 5plex HRM instrument. Finally, the Rotor-Gene AssayManager v2.1 software is used to automatically determine the run/sample validity and calculate the PMR.

Applications

The therascreen PITX2 RGQ PCR Kit enables the determination of the percent methylation ratio (PMR) in promoter 2 of the PITX2 gene. This PMR value of PITX2 promotor 2 is predictive of the response to anthracycline-based chemotherapy regimes in lymph node-positive, estrogen receptor-positive and HER2-negative high-risk breast cancer patients. Applying PITX2 methylation as a predictive biomarker can help clinicians to select the appropriate chemotherapy regimen.

The therascreen PITX2 RGQ PCR Kit is compliant with EU IVD Directive 98/79/EC, enabling the determination of the percent methylation ratio of PITX2 promoter 2 for in vitro diagnostics.

To download Rotor-Gene AssayManager v2.1 after purchase:
Click the "Download" button and follow the on-screen instructions to install Rotor-Gene AssayManager v2.1.
Rotor-Gene AssayManager v2.1 and license must be purchased prior to use.

IMPORTANT: In order to perform and analyze runs, at least one Rotor-Gene AssayManager v2.1 plug-in needs to be installed. Refer to your assay kit handbook for more information.

To purchase a license key for Rotor-Gene AssayManager v2.1:1. After purchasing Rotor-Gene AssayManager v2.1 software and licenses, click the "Download" button and follow the onscreen instructions to install. After successful installation of the Rotor-Gene AssayManager v2.1 and a plug-in, a “key file” will be generated. This "key file" will be attached to the "Technical Support Form" in Step 5, below. 2. Go to the Technical Support Form. Make sure to include all required information and your Rotor-Gene Q or Rotor-Gene Q MDx serial number when filling out the form. 3. For Question 2, "Type of Request", select “Other”.
4. For Question 4, “Your inquiry”, enter "Rotor-Gene AssayManager license request” and include your purchase order number, from your order confirmation. 5. Include the "key file" from Step 1 as an attachment.6. Submit the form. QIAGEN Technical Services will respond via email with a license file within 1–2 working days.
7. After receiving your computer-specific Rotor-Gene AssayManager v2.1 license file, archive the license file locally on your computer.

IMPORTANT: The license file must always exist on the computer that has Rotor-Gene AssayManager v2.1 installed. The license file location should be protected and not subject to change.